The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiationᰔ† Diego Pasini,1 Adrian P

The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiationᰔ† Diego Pasini,1 Adrian P. Bracken,1 Jacob B. Hansen,2 Manuela Capillo,3,4 and Kristian Helin1* Centre for Epigenetics and BRIC, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark1; Department of Biomedical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark2; Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy3; and Institute of Molecular Oncology of the Italian Foundation for Cancer Research, Via Adamello 16, 20139 Milan, Italy4

Received 3 August 2006/Returned for modiﬁcation 10 October 2006/Accepted 22 February 2007 Downloaded from

Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12؊/؊ mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12؊/؊ ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-speciﬁc genes. Moreover, Suz12؊/؊ ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-speciﬁc genes. Finally, we demonstrate that the PcGs are actively recruited to

several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always http://mcb.asm.org/ sufﬁcient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of speciﬁc expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

The evolutionarily conserved Polycomb group (PcG) pro- the molecular mechanisms that control cell fate decisions dur- teins silence gene expression through the formation of multi- ing development. protein complexes. The Polycomb repressive complex 2 Cell fate decisions start taking place before embryo implan- (PRC2) contains the PcG proteins EZH2, EED, and SUZ12 tation, and ICM pluripotency is already lost during embryo and catalyzes the di- and trimethylation of lysine 27 of histone gastrulation (ϳE7.5). Cell type specifications occur through on September 4, 2018 by guest H3 (H3K27me2 and H3K27me3, respectively) (8, 12, 19, 28). It the establishment of specific gene expression programs that has been suggested that this modification serves as a “docking- require epigenetic-dependent transcriptional regulation (2). site” for the PRC1 complex and is required for maintaining Epigenetic control of transcription involves modifications of transcriptional repression (8, 19). The three PcG subunits of both DNA and histones, and the factors that can “write” and PRC2 are all essential for embryonic development, and “read” these modifications play a critical role during develop- Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Eed , Ezh2 , and Suz12 embryos all display severe ment (22). defects during gastrulation (15, 30, 32). SUZ12 is required for Consistent with this model of development, it has not been Ϫ/Ϫ PRC2 enzymatic activity, and Suz12 embryos die around 7 possible to establish Ezh2Ϫ/Ϫ ES cell lines in tissue culture days postcoitus (32). Loss of Suz12 leads to global loss of (30). In contrast, EedϪ/Ϫ ES cells can be expanded in tissue H3K27me3 and to destabilization of Ezh2 (9, 32). These re- culture even though the cells lack global levels of H3K27 meth- sults demonstrate the critical role of Suz12 and PRC2 in reg- ylation (26). EedϪ/Ϫ ES cells have an increased expression of ulating normal development and suggest that PRC2 controls differentiation-specific genes, and the cells “tend” to lose plu- the expression of genes essential for early embryogenesis. ripotency (4). The discrepancy between the phenotypes of Embryonic stem (ES) cells isolated from the inner cell mass Ϫ Ϫ Ϫ Ϫ Ezh2 / and Eed / ES cells suggests that Ezh2 could have (ICM) of preimplantation embryos (embryonic day 3.5 [E3.5]) independent functions that do not involve H3K27 methylation. are immortal and pluripotent. Their self-renewal requires leu- In addition, recent reports have shown that both PRC2 and the kemia inhibitory factor (LIF)-dependent Stat3 activation as PRC1 are required to maintain the repression of differentia- well as the expression of ES cell-specific transcription factors tion-specific genes in mouse and humans ES cells as well as in like Nanog and Oct4 (2). ES cells can give rise to all the somatic cells of an organism and can be differentiated in vitro human embryonic lung fibroblasts (TIG3) (4, 5, 21). to all cell types. They are therefore attractive as a tool to study Here we describe the role of Suz12 in ES cell proliferation and differentiation. We show that Suz12 is essential for proper differentiation, but not proliferation, of ES cells. * Corresponding author. Mailing address: Centre for Epigenetics Importantly, we show that different mechanisms of PcG and BRIC, University of Copenhagen, Ole Maaløes Vej 5, 2200 Co- transcriptional regulation exist during development. This penhagen, Denmark. E-mail: [email protected]. Phone: 45 3532 involves active recruitment of PcGs to repress gene expres- 5666. Fax: 45 3532 5669. E-mail: [email protected]. sion; however, we also demonstrate that increased levels of † Supplemental material for this article may be found at http://mcb .asm.org/. H3K27me3 and PcG binding can also correlate with the ᰔ Published ahead of print on 5 March 2007. activation of gene expression.

3769 3770 PASINI ET AL. MOL.CELL.BIOL.

MATERIALS AND METHODS RESULTS

Derivation of ES cells, culture, genotype, and sex determination. Blastocysts ؊/؊ ϩ Ϫ Establishment of Suz12 ES cells. To study the role of were isolated from the uterus of superovulated pregnant Suz12 / female mice at 3.5 days postcoitus in M16 medium (Sigma). Single blastocysts were cultured Suz12 in ES cell proliferation, we attempted to derive ES cells Ϫ/Ϫ on 0.5% gelatin-coated plates in Glasgow medium (Sigma) supplemented with from the ICM of Suz12 blastocysts. ICM outgrowths iso- glutamine (Gibco), nonessential amino acids (Gibco), sodium pyruvate (Gibco), lated from blastocysts at E3.5 were expanded in tissue culture 50 ␮M ␤-mercaptoethanol–phosphate-buffered saline (PBS; Gibco), and 20% in the presence of LIF. Genotypes of the individual clones ES-cell-tested fetal bovine serum (HyClone) in the presence of 2,000 U/ml of showed that both female and male Suz12Ϫ/Ϫ ES cells could be LIF (ESGRO). ICM outgrowths were expanded and kept in culture in the same Ϫ/Ϫ basal medium with 10% fetal bovine serum and 1,000 U/ml of LIF. Genotyping derived and maintained in culture (Fig. 1A). Suz12 ES cells of single clones was performed as described previously (32). Sex was determined do not display any morphological differences compared to by PCR using the following primers: Xist forward, 5Ј-GCTTTGTTTCACTTTC wild-type and Suz12ϩ/Ϫ cells (Fig. 1A and B, top panels). TCTGGTGC-3Ј; Xist reverse, 5Ј-ATTCTGGACCTATTGGGAAG-GGGC-3Ј; Moreover, the proliferation rate of Suz12Ϫ/Ϫ ES cells did not Ј Ј Ј Sry forward, 5 -GCATTTATGGTGTGGTCCCGTG-3 ; and Sry reverse, 5 -CC differ significantly from wild-type and Suz12ϩ/Ϫ cells (data not Ј Downloaded from AGTCTTGCCTGTATGTGATGG-3 . Ϫ/Ϫ ES cell karyotype and immunostaining. Growing ES cells were treated with shown). Importantly, Suz12 ES cells express normal levels Colticin (10 ␮l/ml; Gibco) for 1 h. Cells were harvested, incubated for 18 min in of the ES cell markers Oct4 and Nanog (Fig. 1C) and have a hypotonic solution (75 mM KCl), and subsequently fixed in fixative solution (1 normal karyotype (Fig. 1D). Consistent with the requirement volume of acetic acid in 3 volumes of methanol). Metaphases were allowed to dry for Suz12 to stabilize Ezh2, Suz12Ϫ/Ϫ ES cells have reduced on slides overnight at 37°C and were stained with DAPI (4Ј,6Ј-diamidino-2- levels of Ezh2 (Fig. 1B). In order to further characterize the phenylindole; Sigma). For immunostaining, ES cells were cultured in normal ES Ϫ/Ϫ cell medium on mitotically inactivated mouse embryonic fibroblasts (MEFs). Suz12 ES cells, we stained for the expression of Oct4 and Cells were fixed for 10 min in 4% buffered formaldehyde and stained with the Nanog. Consistent with the expression data presented in Fig. antibodies indicated in the figure legends in the presence of 10% serum for 1 h 1C, Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells but not feeder cells ex- in a humid chamber. pressed high levels of both Oct4 and Nanog (Fig. 2A and B), http://mcb.asm.org/ Antibodies. Immunoblotting and immunostaining were performed with the Ϫ Ϫ demonstrating that Suz12 / ES cells present normal ES cell following antibodies: rabbit anti-Suz12, anti-H3K27me3, anti-H3K27me1 (where me1 indicates monomethylation), anti-H3K9me3, anti-H3K9me2, and anti- features. H3K4me2 from Upstate; rabbit anti ␤-tubulin from Santa Cruz; rabbit anti- Suz12 is required for di- and trimethylation of H3K27 in ES H3K27me2 (33); mouse anti-EZH2 BD43 (32); mouse anti-H3K27me2/me3 cells. To determine if Suz12 is required for the methylation of (31); and rabbit anti-Nanog and rabbit anti-Oct4 from Abcam. Chromatin im- H3K27 in ES cells, we prepared cell lysates from proliferating munoprecipitation (ChIP) analysis was performed with the following antibodies: ϩ Ϫ Ϫ Ϫ Ϫ Ϫ Suz12 / and Suz12 / ES cells. Remarkably, Suz12 / ES rabbit anti-Suz12 and anti-H3K27me3 from Upstate, rabbit anti-polymerase II and rabbit antihemagglutinin (Y11) from Santa Cruz, mouse monoclonal anti- cells have no detectable H3K27me3 and strongly reduced lev- body to EZH2 (AC22) (32), mouse monoclonal antibody to BMI1 (AF27) (5), els of H3K27me2, whereas the methylation of other lysines on and two rabbit affinity-purified polyclonal antibodies to CBX8 (hPc3), LAST and the histone tails remains unchanged (Fig. 1E). These results on September 4, 2018 by guest GALD (5). suggest that PRC2 is the major (if not the only) histone methyl Gene expression analysis. Total RNA was extracted independently from three Suz12ϩ/Ϫ (clones SBE 4, SBE5, and SBE6) and three Suz12Ϫ/Ϫ (clones SBE1, transferase responsible for H3K27me3 in ES cells. In addition, SBE7, and SBE8) ES cell clones. RNA was quantified, and equal amounts from they show that neither Suz12 nor of H3K27me is essential for the three Suz12ϩ/Ϫ and the three Suz12Ϫ/Ϫ samples were pooled into one sample the self-renewal of ES cells. -to reduce the experimental variation. Targets for microarray hybridization were Suz12؊/؊ ES cells express higher levels of differentiation synthesized according to the supplier’s instructions (Affymetrix). Hybridization, specific genes. To further analyze the features of Suz12Ϫ/Ϫ ES washing, staining, scanning, and data analysis were performed at the Affymetrix microarray unit at the Institute of Molecular Oncology of the Italian Foundation cells, we compared the global gene expression profiles of Ϫ/Ϫ ϩ/Ϫ for Cancer Research-European Institute of Oncology campus, Milan, Italy, ac- Suz12 and Suz12 ES cells using Affymetrix oligonucle- cording to the manufacturer’s instructions. Expression levels were analyzed using otide microarrays containing probes for more than 39,000 dif- Microarray Analysis Suite, version 5.0, statistical algorithm software (Af- ferent mouse transcripts. Significantly, Suz12Ϫ/Ϫ ES cells con- fymetrix), using the default parameters and scaling (TGT value) signal intensities ϩ/Ϫ tain increased levels of differentiation-specific genes, for all the GeneChip arrays to a value of 500. The Suz12 samples were used Ϫ/Ϫ as a baseline condition for comparison with the Suz12Ϫ/Ϫ samples. suggesting that Suz12 ES cells have differentiated features, Quantitative PCR and primers. cDNA preparation and real-time quantitative despite displaying a normal stem cell phenotype (Fig. 3A). To PCR (qPCR) were performed following the manufacturer’s instructions (Ap- validate the gene expression data, we determined the expres- plied Biosystems). The analysis of the results was performed as described pre- sion levels for 25 upregulated and 5 downregulated genes in viously (32). For primer sequences see Table S2 in the supplemental material. Suz12Ϫ/Ϫ and Suz12ϩ/Ϫ ES cells by real-time qPCR. As shown EB formation and neuronal differentiation. Embryoid bodies (EBs) were allowed to form in the absence of LIF in hanging drops containing 1,000 ES in Fig. S1A in the supplemental material, the microarray data cells/20-␮l drop on petri dish lids for 48 h. EBs were collected from the drops were confirmed for all the tested genes, demonstrating the high after 2 days and left in culture in noncoated petri dishes for the times indicated accuracy of the expression profile data. Because these genes in the figure legends in ES medium in the absence of LIF. Medium was changed could be potential direct targets of the PRC2 complex, we every 2 days. Neuronal differentiation followed the above EB formation protocol tested Ezh2 and Suz12 binding and the presence of H3K27me3 with the following modifications: EBs were treated from day 2 to day 5 with 0.5 ␮M all-trans-retinoic acid (ATRA) and plated on gelatin-coated dishes at day 7 modification within a 3-kb promoter region upstream of the to allow neuronal differentiation. For ChIP analysis EBs were formed as mass transcription start site of the genes. ChIP analysis revealed that cultures by plating ES cells in suspension on noncoated petri dishes at the 10% (3/30) of the analyzed genes were direct targets of PRC2 concentration of 5 ϫ 105 cells/ml. (see Fig. S1 in the supplemental material) and that Ezh2 bind- ChIP. ChIP assays were performed as described previously (6). ing and accumulation of H3K27me3 at the Tbx3, Satb2, and Animal studies. Animal care and experiments on live animals were performed at the University of Copenhagen in accordance with the Danish institutional and Otx2 promoters are dependent on Suz12 (see Fig. S1 in the national guidelines (law number 726, 9 September 1993), and the studies were supplemental material). These results are consistent with the approved by the Dyreforsøgstilsynet committee (project number 2004/561-860). requirement of Suz12 for Ezh2 enzymatic activity. VOL. 27, 2007 Suz12 IS REQUIRED FOR ES CELL DIFFERENTIATION 3771 Downloaded from http://mcb.asm.org/ on September 4, 2018 by guest

FIG. 1. Analysis of Suz12Ϫ/Ϫ ES cells. (A) Phase-contrast pictures of growing Suz12ϩ/Ϫ (clone SBE4) and Suz12Ϫ/Ϫ (SBE8 8) ES cell clones growing on feeders cells. (B) Phase-contrast pictures (top) of growing Suz12ϩ/Ϫ (clone SBE4) and Suz12Ϫ/Ϫ (SBE1 and SBE8) ES cell clones on gelatin-coated plates. Genotype and sex determination PCRs (middle) show that both male (M) and female (F) Suz12Ϫ/Ϫ ES cells can be derived. Immunoblots (bottom) using antibodies to Suz12, Ezh2, and ␤-tubulin are shown. ␤-Tubulin served as a loading control. KO, knockout; WT, wild type. (C) RNA expression levels of the ES cell markers Oct4 and Nanog in Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES clones compared to MEFs. (D) Metaphase spreads showing a normal karyotype for Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES clones. At the top the average numbers of counted chromosomes per cell are given. The bottom panels are representative pictures of metaphase spreads from Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES clones. (E) Immunoblots of different histone H3 lysine (K) modifications using specific antibodies to the indicated proteins and their modifications.

In agreement with our results, Tbx3, Satb2, and Otx2 were sults are consistent with the model by which PRC2 represses identified as direct PRC2 targets in whole genomic screenings the expression of differentiation-specific genes in ES cells (4, aimed at identifying PRC targets genes in ES cells (4, 21). 21) and demonstrate that Suz12 is required for this activity. Surprisingly, several of the other PRC2 target genes identified Interestingly, we also found that the expression of the pa- in previous studies were not identified as upregulated in ternally imprinted H19 gene is significantly increased in Suz12Ϫ/Ϫ ES cells. One explanation for this could be that the Suz12Ϫ/Ϫ ES cells relative to expression in to Suz12ϩ/Ϫ ES cells expression of these genes is not detectable in Suz12ϩ/Ϫ ES cells (Fig. 3C; see also Table S1 and Fig. S1A in the supplemental by microarray technology, and they were therefore discarded in material). This result is consistent with the requirement of Eed the statistical analysis. To address this possibility, we compared for the imprinting of different autosomal loci (24), suggesting the expression of three differentiation-specific transcription that imprinting could also be affected in Suz12Ϫ/Ϫ embryos. To factors, Gata1, Gata4, and Hnf4,inSuz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES understand if the PRC2 complex and H3K27me3 are directly cells. As shown in Fig. 3B, Gata1, Gata4, and Hnf4 expression involved in the maintenance of H19 repression, we analyzed was indeed higher in the Suz12Ϫ/Ϫ ES cells than in Suz12ϩ/Ϫ the binding of Ezh2 and Suz12 and the presence of histone cells. Consistent with the repression by PRC2, the H3K27me3 H3K27me3 modification both at the H19 promoter (Ϫ5toϩ1 repressive marker was present at the Gata1, Gata4, and Hnf4 kb with respect to the transcription start site) and at the CG promoters and lost in Suz12Ϫ/Ϫ ES cells (Fig. 3B). These re- regulatory element placed at the 3Ј end of the H19 locus. ChIP 3772 PASINI ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/ on September 4, 2018 by guest

FIG. 2. Expression of Nanog and Oct4 in Suz12Ϫ/Ϫ ES cells. Immunostaining of Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells growing on feeders cells shown with anti-Nanog-speciﬁc (A) and anti-Oct4-speciﬁc (B) antibodies reveals expression of both ES cell markers in Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells but not in feeder cells (arrows).

analysis showed that neither PRC2 nor H3K27me3 was present When ES cells are cultured in suspension in the absence of in the analyzed genomic regions (see Fig. S1A in the supple- LIF, they form EBs in which cells differentiate into the three mental material), suggesting that either the PRC2 regulation is germ layers. EBs are the in vitro developmental equivalent of indirect or that it involves the recruitment of PRC2 activity at the mouse embryo at the egg cylinder stage (E5.5 to E6). They different sites or in earlier developmental stages. contain an outer endoderm layer and differentiating cells in the Suz12 is required for differentiation of ES cells. Our findings center of the body, together with epithelial-like cavities. Be- are consistent with the fact that Suz12Ϫ/Ϫ, Ezh2Ϫ/Ϫ, and cause EBs represent the developmental stage when PRC2 mu- EedϪ/Ϫ embryos are all able to implant but fail to undergo tant embryos display morphological abnormalities and die, we further development, and they suggest that PRC2 and its en- investigated whether EBs derived from Suz12Ϫ/Ϫ ES cells zymatic activity are required for the differentiation processes present any defects in differentiation. As shown in Fig. 4C and taking place during development. To investigate this, we took Fig. S2C in the supplemental material, EBs from Suz12ϩ/Ϫ advantage of the fact that ES cells undergo neuronal differen- cells are morphologically normal, presenting an endodermal tiation in the presence of ATRA. Thus, we treated Suz12ϩ/Ϫ outer layer and organized epithelial structures (Fig. 4C, left and Suz12Ϫ/Ϫ ES cells with ATRA and found that while frames). In contrast, Suz12Ϫ/Ϫ EBs display a disorganized Suz12ϩ/Ϫ ES cells efficiently form neurons, we were unable to structure and are often smaller. In addition, Suz12Ϫ/Ϫ EBs fail detect any neurons in ATRA-treated Suz12Ϫ/Ϫ ES cells (Fig. to form a proper endodermal layer and lack any form of in- 4A; see also Fig. S2A in the supplemental material). Consis- ternal organized structures (Fig. 4C, right frames; see also Fig. tent with this, the expression of two neuron-specific markers, S2C in the supplemental material). Consistent with the ability GluR6 and Gad65, was strongly activated in Suz12ϩ/Ϫ differ- of Suz12Ϫ/Ϫ ES cells to form EBs in vitro, Suz12Ϫ/Ϫ ES cells entiated cells, but not in Suz12Ϫ/Ϫ cells (Fig. 4B; see also Fig. were able to form teratomas when subcutaneously injected into S2B in the supplemental material). immunodeficient mice (data not shown). The differentiation of ES cells in tissue culture first requires To further support these results, we compared the expres- the formation of EBs, followed by terminal differentiation (18). sion of specific ES cell markers as well as the expression of VOL. 27, 2007 Suz12 IS REQUIRED FOR ES CELL DIFFERENTIATION 3773 Downloaded from http://mcb.asm.org/ on September 4, 2018 by guest

FIG. 3. Suz12 is required for the regulation of a large number of genes involved in development, differentiation, and homeostasis. (A) Func- tional clustering of gene expression changes between Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells. Expression downregulation refers to the functional clustering of genes whose expression was downregulated in Suz12Ϫ/Ϫ compared to Suz12ϩ/Ϫ ES cells. Expression upregulation refers to the functional clustering of genes whose expression was upregulated in Suz12Ϫ/Ϫ compared to Suz12ϩ/Ϫ ES cells. (B) Expression (top) and ChIP analysis using the indicated antibodies (bottom) were determined by real-time qPCR. HA, hemagglutinin. (C) Expression levels of H19 in Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ male ES clones.

genes specifically activated during gastrulation in Suz12ϩ/Ϫ the expression of Oct4 and Nanog was fully repressed during and Suz12Ϫ/Ϫ differentiating ES cells. Consistent with the lack ES cell differentiation in Suz12ϩ/Ϫ ES cells, whereas the ex- of germ layer formation, the expression of gastrulation-specific pression of the two stem cell transcription factors was only genes (Brachyury, Pax3, Pax7, Fgf3, and Wnt3a) was activated partly repressed in Suz12Ϫ/Ϫ EBs (Fig. 5A; see Fig. S2D in the only in Suz12ϩ/Ϫ and not Suz12Ϫ/Ϫ EBs (Fig. 5B). Moreover, supplemental material), with expression levels approximately 3774 PASINI ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/ on September 4, 2018 by guest FIG. 4. Suz12Ϫ/Ϫ ES cells fail to undergo proper differentiation. (A) Phase-contrast pictures of neuronal differentiation of Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells. Arrows in the left panels show neuron formation in Suz12ϩ/Ϫ cells, while the right panels show lack of neuron formation in Suz12Ϫ/Ϫ cells. (B) Expression levels of two specific neuronal markers GluR6 and Gad65 showing strong activation in differentiated Suz12ϩ/Ϫ cells and no activation in Suz12Ϫ/Ϫ cells. Undiff, undifferentiated; diff, differentiated. (C) Hematoxylin and eosin staining of 7-day EBs formed by Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells. Top panels show normal morphology of Suz12ϩ/Ϫ EBs. High-magnification fields highlight outer endodermal layers and epithelium-like cavities. Bottom panels show Suz12Ϫ/Ϫ EBs that lack forms of organized structures and that are often smaller.

50-fold higher in Suz12Ϫ/Ϫ EBs at 9 days of differentiation entiation and that the lack of Suz12 results in both deregula- (Fig. 5A, right). Western blots presented in Fig. 5C further con- tion of ES cell-specific genes and the inactivation of genes firm this result and show that Oct4 and Nanog protein levels are required for early embryogenesis. indeed still expressed in Suz12Ϫ/Ϫ differentiated ES cells but not Mechanisms for PRC-mediated transcriptional regulation in the control cells. Similar expression results were also obtained during differentiation. Recently, we have identified genes tar- for other genes like Fgf4, Fgf17, and Pou2F3 that are expressed in geted by PRC2 and PRC1 in a human diploid fibroblast cell ES cells and repressed upon differentiation (Fig. 4A). line, TIG3 (5). Interestingly, the genes presented in Fig. 5A In addition, due to the inability of Suz12Ϫ/Ϫ ES cells to and B were identified as direct targets of both PRCs in TIG3 terminally differentiate into neurons, we tested whether cells, suggesting that the PRCs remain associated with the Suz12Ϫ/Ϫ ES cells are also impaired in the ability to differen- promoters after their recruitment during early embryogenesis. tiate into early neuronal precursors. We analyzed the expres- To determine if the binding of the PRCs is conserved between sion of the early neuronal markers Sox1, Nestin, Musashi, and human and mouse, we performed ChIP analysis in MEFs (see Calb2 during Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cell differentiation. Fig. S3A and B in the supplemental material). While Ezh2, Consistent with the lack of terminal differentiation, the activa- Suz12, and histone H3K27me3 were present on all promoters, tion of the expression of Sox1, Nestin, Musashi, and Calb2 the PRC1 proteins Cbx8 and Bmi1 were not. Significant Cbx8 during ES cell differentiation was observed in only Suz12ϩ/Ϫ binding was detected at the Brachyury, Pax3, Pax7, Fgf4, and and not Suz12Ϫ/Ϫ ES cells. This result showed that Suz12Ϫ/Ϫ Oct4 promoters, while significant Bmi1 binding was detected at ES cells are impaired in the proper activation of the expression all promoters except for the Pou2f3. The differential binding of neuronal precursor markers and suggest that the differen- could be due to cell type differences and suggests that the tiation defects of Suz12Ϫ/Ϫ ES cells occur during the early composition of PRC1 may differ from one cell type to another. commitment phase of differentiation. Taken together, these However, the results show that PRC2 binding at these genes is results demonstrate that Suz12 is required for ES cell differ- fully conserved between mouse and human. VOL. 27, 2007 Suz12 IS REQUIRED FOR ES CELL DIFFERENTIATION 3775 Downloaded from http://mcb.asm.org/ on September 4, 2018 by guest

FIG. 5. Suz12Ϫ/Ϫ ES cells fail to repress ES cells markers and to activate differentiation-speciﬁc genes upon induction of differentiation. (A) Expression levels of Oct4, Nanog, Fgf4, Fgf17, and Pou2f3 in ES cells and in 9-day differentiated EBs determined by real-time qPCR. Right panels highlight the expression differences between Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ in 9-day differentiated EBs. (B) Expression levels of gastrulation markers in ES cells and in EBs at 3, 6, and 9 days after induction of differentiation. (C) Immunoblotting for Oct4 and Nanog during Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cell differentiation showing repression of Oct4 and Nanog expression in Suz12ϩ/Ϫ but not Suz12Ϫ/Ϫ ES cells. (D) Expression levels of Sox1, Nestin, Mausashi, and Calib2 during Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cell differentiation showing the lack of activation of the neuronal precursor marker in Suz12Ϫ/Ϫ cells.

Interestingly, while Brachyury, Pax3, Pax7, Wnt3A, and Fgf3 differentiation. Surprisingly, we found that even though these are expressed at low levels in ES cells and become activated two groups of genes are inversely expressed during ES cell upon differentiation (Fig. 4 and 5B), Fgf4, Pou2F3, Fgf17, differentiation (Fig. 5A and B), increased amounts of Nanog, and Oct4 are expressed in ES cells and become re- H3K27me3 associate with all the promoters in a Suz12-depen- pressed upon differentiation (Fig. 5A). Nanog and Oct4 are dent manner during ES cell differentiation (Fig. 6 and 7A). In essential transcription factors for ES cell self-renewal (17, 25) fact, consistent with the requirement of Suz12 for Ezh2 his- and function both as activators and repressors of important tone methyl transferase activity, Ezh2 binding and developmental regulators (3, 23). Pou2f3 is a member of the H3K27me3 association were abolished in Suz12Ϫ/Ϫ differ- Oct transcription factor family, but its biological role is poorly entiated ES cells (Fig. 6 and 7A). This result demonstrates characterized. Instead, Fgf4 and Fgf17 function as signaling that increased association of histone H3K27me3 does not molecules that play a crucial role in the control of develop- prevent transcription and indicates that this modiﬁcation ment. Fgf4, for example, is required for the proliferation of could have a role in transcriptional activation. Despite the trophoblast stem cells (14). binding of PRC2 to the Oct4 and Nanog promoters in MEFs To start addressing the mechanisms regulating the expres- (see Fig. S3 in the supplemental material), we were unable sion of the PcG target genes during early differentiation, we to detect any signiﬁcant PRC2 binding and H3K27me3 as- analyzed the recruitment of Ezh2, Suz12, and H3K27me3 in sociation to these promoters during ES cell differentiation both Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells induced to undergo (see Fig. S4 in the supplemental material), suggesting that 3776 PASINI ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/

FIG. 6. PcG binding does not prevent transcriptional activation. ChIP analyses performed on promoters of genes that are activated during differentiation of ES cells. Real-time qPCR was used to determine the expression levels of the genes, and values were normalized as described in Materials and Methods. Antibodies speciﬁc for Ezh2, Suz12, Cbx8, Bmi1, H3K27me3, and the hemagglutinin (HA) epitope (negative control) were on September 4, 2018 by guest used for ChIPs. Enrichment is given as a percentage of input. Black bars, Suz12ϩ/Ϫ cells; red bars, Suz12Ϫ/Ϫ cells; d, day; Ab, antibody.

PRC2 indirectly regulates these promoters during the early mulates during differentiation (Fig. 7B). Interestingly, Cbx8 events of ES cell differentiation. expression is not fully induced in Suz12Ϫ/Ϫ differentiated ES To understand if PRC1 is also recruited to these PRC2 cells, which could be a result of the lack of proper differenti- target promoters, we analyzed the binding of two subunits of ation of these cells. Unlike Cbx8, Bmi1 is expressed in ES cells PRC1, Cbx8 and Bmi1, in Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells. and further accumulates 3 days after induction of differentia- Consistent with the idea that both PRC1 and PRC2 are re- tion. However, since Bmi1 is not recruited to PRC1 target quired for the regulation of the expression of common targets genes in nondifferentiated cells, this may suggest that Bmi1 (4, 5, 21), we found that five of these promoters (Brachyury, recruitment is dependent on Cbx8 expression. In addition, Pax3, Pax7, Fgf3, and Fgf4) showed significant binding of Cbx8 slower-migrating forms, which might result from phosphoryla- in differentiated EBs, while only one promoter (Pax7) showed tions, as suggested by previously published findings, (35), ap- binding of Bmi1 (Fig. 6 and 7A). However, in contrast to the pear during the differentiation of the ES cells with kinetics model that has PRC1 recruitment dependent on its ability to similar to Cbx8 accumulation. These forms become prominent specifically bind H3K27me3 (7, 8, 19), we found that PRC1 in MEFs, which may suggest that posttranslation modifications recruitment is independent of H3K27me3 on three of the five of Bmi1 could be a regulatory mechanism for the recruitment Cbx8 targets (Pax7, Fgf3, and Fgf4) and on the Bmi1 target of PRC1 to target genes. (Pax7). This result, demonstrates that PRC1 can be recruited to target genes in the absence of a functional PRC2 complex DISCUSSION and independently of H3K27me3. Interestingly, Cbx8 and Bmi1 are not present on PcG target The development of the mammalian organism requires the genes in nondifferentiated ES cells (Fig. 6 and 7A and data not specification of more than 200 different cell types in a process shown). To obtain a potential explanation for this, we prepared that takes place prior to and immediately after the implanta- cell lysates from Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells at different tion of the embryo. Cell type specification is controlled by stages of differentiation, and from proliferating (passage 3 gradients of tissue-specific transcription factors in concert with [P3]) and senescent (P7) MEFs (Fig. 5B). Remarkably, Cbx8 is a host of epigenetic regulators. Histone modifying enzymes, not detectably expressed in undifferentiated ES cells but accu- like the PRCs, are believed to play a critical role in this pro- VOL. 27, 2007 Suz12 IS REQUIRED FOR ES CELL DIFFERENTIATION 3777 Downloaded from http://mcb.asm.org/ on September 4, 2018 by guest

FIG. 7. PRC2 is actively recruited to repress gene transcription during ES cell differentiation. (A) ChIP analysis of the promoter regions of repressed genes during ES cell differentiation using antibodies against Ezh2, Suz12, Cbx8, Bmi1, and histone H3K27me3. Results of ChIPs and qPRCs in Suz12ϩ/Ϫ (black bars) and Suz12Ϫ/Ϫ ES cells (red bars) are shown. The expression profile for each gene during differentiation is also presented (far left). HA, hemagglutinin; Ab, antibody. (B) Immunoblots of Suz12, Ezh2, Eed, Cbx8, and Bmi1 in Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells and in proliferating (P3) and senescent (P7) MEFs. ␤-Tubulin served as a loading control. (C) Different models for how the PcG proteins regulate transcription during differentiation. In the derepression model (1) the PcGs repress the expression of differentiation-specific genes in proliferating ES cells. The loss of PcG binding during differentiation leads to the activation or derepression of transcription. In the repression model (2) the PcGs are specifically recruited to target genes that undergo transcriptional repression during differentiation. In the activation model (3) PcGs (PRC2) accumulate on a subset of target genes during differentiation despite their transcriptional activation. In this model we propose that the binding of transcriptional activators is sufficient to overcome the PcGs. The binding of the PcGs could be important for the repression of the target genes during terminal differentiation and in this way preprogram the target genes during early development. 3778 PASINI ET AL. MOL.CELL.BIOL. cess. PRC2 is required for correct expression of the HOX genes ES cell differentiation, our analysis of PcG target genes sug- in later development (1, 20, 34), but defects in embryos lacking gests a number of mechanisms by which the PcGs control functional PRC2 arise earlier than HOX expression, suggesting transcription. So far the only mechanism that is backed up by additional functions of the complex (16, 30, 32). experimental data is outlined in the derepression model (Fig. Ϫ Ϫ In this work we have shown that even though Suz12 / ES 7C, model 1). This model involves the dissociation of PcG cells proliferate and appear normal, their ability to undergo proteins upon induction of differentiation and allows activation proper differentiation is compromised. We provide evidence of target genes and subsequent differentiation. Several exper- that this is due to the failure to establish the correct transcrip- imental results using myoblasts (10), ES cells (4, 21), and tion programs during early embryogenesis. This assumption is NTera2 cells (5) support this model. ChIP analysis presented Ϫ/Ϫ in agreement both with the fact that Suz12 and Eed mutant in this work further supports the derepression model showing ES cells expression patterns are altered toward a more differ- that differentiation-specific genes are indeed repressed by the entiated phenotype and with the finding that Ezh2 activity is PcGs in Suz12ϩ/Ϫ ES cells and that this repressive activity is required for correct development before the four-cell stage lost in Suz12Ϫ/Ϫ ES cells. Downloaded from Ϫ/Ϫ during embryogenesis (4, 13). Similarly to Suz12 ES cells, In this study, we have shown that the PcGs are also actively Eed mutant ES cells can be maintained in culture, and, despite recruited to target genes upon induction of differentiation. their tendency to differentiate (4), they can efficiently form EBs Surprisingly, we found that this recruitment can correlate with in vitro and contribute to gastrulation in vivo in chimeric em- either transcriptional repression or activation of genes during bryos (27). Interestingly, the fact that Ezh2 (30), but not Eed differentiation. and Suz12, is required for ES cell proliferation indicates that In the first case, we found that differentiation signals lead to Ezh2 has additional functions independent of H3K27 methyl- recruitment of PRC2 to target genes and that this recruitment ation. This notion is supported by data obtained in Drosophila, correlates with repression of transcription. Importantly, this http://mcb.asm.org/ where mutants of the Ezh2 homologue E(z) have additional repression is dependent on Suz12, suggesting that direct bind- phenotypic defects to those observed in Esc (dEed) and ing of PRC2 plays a critical role in the repression of these Su(z)12 (dSuz12) mutant flies (1, 20, 34). genes. Based on these results we propose a mechanism, sum- The effects of the lack of PRC2 transcriptional control are marized in the repression model presented in Fig. 7C, whereby more evident when Suz12Ϫ/Ϫ ES cells are induced to differen- the PcGs regulate transcription of specific target genes during tiate. We found that Suz12 is required for proper ES cell differentiation. If this model is accurate, the identification of differentiation. Key developmental transcription factors such the signaling pathways and the factors that trigger the specific as Brachyury, Pax3, and Pax7 as well as early neuronal markers recruitment of the PRCs will be important for understanding such as Sox1, Nestin, Musashi, and Calb2 are not activated by how differentiation is regulated. differentiation signals in the absence of Suz12. Moreover, dur- on September 4, 2018 by guest In the second case, we found increased levels of PRC2 on ing differentiation Suz12 appears to be required for the repres- the promoters of genes activated during differentiation. Since sion of genes essential for ES cell self-renewal, such as Nanog PRC2 is believed to function as a repressor of transcription, and Oct4. This could be a consequence of the differentiation Ϫ/Ϫ this is an intriguing result. Furthermore, the striking observa- failure of the Suz12 ES cells, but since ectopic expression of Ϫ/Ϫ Nanog can block ES cell differentiation in vitro (11), sustained tion that the genes were not activated in Suz12 ES cells, i.e., Nanog expression in the Suz12Ϫ/Ϫ ES cells could also contrib- in the absence of PRC2 recruitment, could suggest that PRC2 ute to the impairment of proper differentiation of the Suz12Ϫ/Ϫ has a direct role in the transcriptional activation of these tar- ES cells. Our results showing that PcGs bind directly to the gets. However, it is important to stress that the lack of tran- Nanog and Oct4 promoters in MEFs and that they are associ- scriptional activation could be a consequence of the failure of Ϫ/Ϫ ated with H3K27me3 support the notion that the expression of Suz12 ES cells to undergo proper differentiation. In this these promoters is controlled by the PcGs. In any event, it case, the early recruitment of the PRCs could simply epige- would be extremely interesting to know if the differentiation netically predispose these genes for later repression. A model defects of Suz12Ϫ/Ϫ ES cells depend on lack of Nanog and/or for how PcGs may participate in the activation of target genes Oct4 repression; however, this experiment is unfortunately not is depicted in Fig. 7C. feasible because ES cells do not grow in the absence of the two In conclusion, our results and those from other laboratories transcription factors (25, 29). demonstrate that Suz12 and PRC2 are required for ES cell Our results demonstrating the accumulation of the PRCs differentiation, most likely by directly controlling specific gene and H3K27me3 on promoters for differentiation-induced expression during cellular commitment. ES cells become an genes is very exciting and support our previous findings that attractive tool for regenerative therapy due to their in vitro PcG-binding and H3K27me3 accumulation might not be suf- differentiation potential. Embryo cloning by somatic cell nu- ficient to prevent transcription (5). In our previous work (5), clear transfer (SCNT) could give important therapeutic advan- we proposed that PcG binding to active promoters in progen- tages as a source of ES-like cells that can be in vitro differen- itor cells preprograms these genes for repression during ter- tiated and used for autologous implants. Low efficiency in minal differentiation. Here, we have provided evidence that therapeutic cloning is the major obstacle to overcome because the PcGs are indeed specifically recruited to active promoters epigenetic commitment of somatic cells is a major problem in in progenitor cells. Moreover, our results could further suggest successful embryo cloning. The failure to achieve correct re- that the recruitment of the PcGs to these target genes may be activation of genes like Nanog and Oct4 has been linked to the required for their transcriptional activation. failure of embryos obtained by SCNT to implant and success- In addition to demonstrating the essential role of Suz12 in fully develop. Manipulation of PRC2 activity could therefore VOL. 27, 2007 Suz12 IS REQUIRED FOR ES CELL DIFFERENTIATION 3779 contribute to the reprogramming of somatic cells and increase 15. Faust, C., K. A. Lawson, N. J. Schork, B. Thiel, and T. Magnuson. 1998. The the efficiency of successful SCNT embryo cloning. Polycomb-group gene eed is required for normal morphogenetic movements during gastrulation in the mouse embryo. Development 125:4495–4506. 16. Faust, C., A. Schumacher, B. Holdener, and T. Magnuson. 1995. The eed ACKNOWLEDGMENTS mutation disrupts anterior mesoderm production in mice. Development 121: 273–285. We thank Elena Prosperini, Daniele Piccini, and Micaela Quarto for 17. Hay, D. C., L. Sutherland, J. Clark, and T. Burdon. 2004. Oct-4 knockdown technical assistance and Alberto Gobbi for suggestions. We thank induces similar patterns of endoderm and trophoblast differentiation mark- Thomas Jenuwein for providing the H3K27me2 antibody, Danny ers in human and mouse embryonic stem cells. Stem Cells 22:225–235. Reinberg for the H3K27me2/me3 antibody, and Klaus Hansen for the 18. Keller, G. M. 1995. In vitro differentiation of embryonic stem cells. Curr. CBX8 antibodies. We thank Emmanuelle Trinh, Anders H. Lund, Opin. Cell Biol. 7:862–869. Michael Lees, and Luciano Di Croce for critical reading of the manu- 19. Kuzmichev, A., K. Nishioka, H. Erdjument-Bromage, P. Tempst, and D. script. We thank members of the Helin laboratory for technical advice Reinberg. 2002. Histone methyltransferase activity associated with a human and support. multiprotein complex containing the Enhancer of Zeste protein. Genes Dev. 16:2893–2905. This work was supported by grants from the Association for Inter- 20. LaJeunesse, D., and A. Shearn. 1996. E(z): a polycomb group gene or a national Cancer Research, the Danish Cancer Society, the Danish trithorax group gene? Development 122:2189–2197. Downloaded from Medical Research Council, the Danish Natural Science Research 21. Lee, T. I., R. G. Jenner, L. A. Boyer, M. G. Guenther, S. S. Levine, R. M. Council, and the Danish National Research Foundation. J.B.H. was Kumar, B. Chevalier, S. E. Johnstone, M. F. Cole, K. Isono, H. Koseki, supported by grants from the Carlsberg Foundation and the Novo T. Fuchikami, K. Abe, H. L. Murray, J. P. Zucker, B. Yuan, G. W. Bell, E. Nordisk Foundation. Herbolsheimer, N. M. Hannett, K. Sun, D. T. Odom, A. P. Otte, T. L. Volkert, D. P. Bartel, D. A. Melton, D. K. Gifford, R. 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